Each cancer patient is unique, as is the tumor and the cancer. The course of the disease and the effect of therapy are a product of the genetics and the environment of both the patient and the tumor. Through tailoring of chemotherapy to the specific genetic profile of the patient and the tumor, pharmacogenetics may potentially increase the efficacy, lessen the toxicity and decrease the overall costs of chemotherapy. At the cellular level cancer is a genetic disease and progression of the tumor is associated with a progressively larger number of genetic changes, e.g. translocations, amplifications, deletions and mutations. The genes involved in the cancerous process are oncogenes and tumor suppressor genes. Oncogenes are activated through translocations and amplifications while tumor suppressor genes are eliminated primarily through mutation and deletions.
The Burden of Cancer
Based on 1998-2000 data from the Surveillance, Epidemiology, and End Results (SEER) program it have been demonstrated that the lifetime risk of developing cancer is 1 in 3 for females and 1 in 2 for males (Http://www.seer.cancer.gov). The 5-year relative survival rate varies considerably by cancer site and stage at diagnosis. Significant decreases in death rates have been observed for cancer at all sites combined, for female breast cancer and colorectal cancer of both sexes. The three most frequent cancers occurring in females are breast, lung and colon cancer, compared to males where prostate, lung and colon cancer are the most frequent. It can also be estimated that close to 10 million people in the U.S. are alive after being diagnosed with cancer. Over 2 million women who have had a previous diagnosis of breast cancer are alive today, reflecting both the high incidence and the relatively good survival of breast cancer.
The Contribution of Cytotoxic Chemotherapy to Survival of Cancer Patients
Treatment with cytotoxic agents is a well-established standard for most advanced cancers and is also increasingly becoming an integrated part of treatment in primary and early cancers, examples being colorectal and breast cancers. Cytotoxic chemotherapy can in early cancers be given before surgery and radiotherapy as the primary treatment, concomitant with local therapy or adjuvant to local therapies. DNA synthesis inhibitors as 5-Fluorouracil (5-FU) have demonstrated activity in a range of advanced cancers, including colorectal, breast, and head & neck cancers. The greatest impact is however achieved in patients with operable colorectal and breast cancer were treatment with 5-FU containing chemotherapy significantly reduces the risk of recurrence and mortality (IMPACT Lancet 1995; 345:939-44, EBCTCG Lancet 2005).
The Example of Breast Cancer
Patients with primary breast cancer will often receive a complex therapy integrating multiple modalities. The local therapy may be surgery alone or a combination of surgery and radiotherapy. Systemic therapies will be advised to the majority of patients depending on characteristics of the patient and the tumor. Survival is at least as good with pre- as with postoperative chemotherapy, but the different treatment modalities are primarily given in sequence to avoid a possible interaction between different modalities.
The somatic genetic alterations involved in the etiology of breast cancer are complex and heterogeneous. Several proto-oncogenes known to be activated include MYC, ERBB2 (HER2), and CCND1 [S Ingvarsson 1999]. Activation of these genes often involves genetic aberrations such as e.g. amplification and/or translocation of the corresponding chromosomal regions, and e.g. mutations and deletions of tumor suppressor genes, such as TP53, CDH1 (encoding E-cadherin) and FHIT, have also been demonstrated. ERBB2 over-expression is observed in 20-30% of breast cancers and in general ERBB2 over expression is associated with poor prognosis. However, there are also data from retrospective studies indicating that the ERBB2 status might be of predictive value as well. TP53 mutation is observed in about 25% of breast cancers. Studies evaluating the prognostic (and predictive) value of p53 using immunohistochemical methods to determine p53 status are conflicting. However, detection of TP53 gene alterations is superior to immunostaining as a prognostic factor [S Thorlacius 1995] and TP53 mutation shows a strong association with poor prognosis [J Overgaard 2000]. It appears that TP53 mutation, or at least a subset of mutations, is also associated with resistance to anthracycline-based chemotherapy [S Geisler 2001].
Although much effort has been devoted to the study of predictive markers in cancer, only very limited details have emerged until now. In breast cancer the only predictive markers in clinical use are ER, PgR and ERBB2, predicting sensitivity to hormone and Herceptin treatment, respectively. Additional markers are in preparation for a clinical use, e.g. TOP2A copy number changes (deletion or amplification) as predictive to the outcome of anthracycline treatment. However, given the wide range of chemotherapy regimens used there is a huge need of more predictive markers to facilitate the introduction of personalized medicine.
Predictive Factors for Efficacy of Adjuvant Chemotherapy in Breast Cancer.
The efficacy of chemotherapy is well documented in early breast cancer [EBCTCG, Lancet 2005]. The first generation of clinical trials showed that chemotherapy e.g. the classical combination of Cyclophosphamid, Methotrexate and Fluorouracil (CMF) reduces the risk of recurrence and mortality of breast cancer with 20-25%. Further risk reduction has been achieved in later trials adding anthracyclines, and currently the efficacy of taxanes is being investigated. Chemotherapy is offered to a prognostic heterogeneous group of patients where the individual risk of recurrence and death from breast cancer varies from 10% to 80%. Totally, 30-40% of the expected deaths can be prevented, but in total numbers the overall survival is only reduced with a few percent (from 10% to 7%) in the low risk group, and with approximately 25% in the high-risk group (from 80% to 55%). Thus, extensive over treatment takes place with concurrent consequences in form of side effects and unintended use of resources. Therefore, a high need of development of predictive factors is present and until now unmet.
Often the mechanism of action is not known in details when a treatment is introduced in the clinical setting. A starting insight into the molecular background of the mode of action of some chemotherapeutics is emerging, and recently some examples of a relationship between the effect of treatment with a specific chemotherapy and specific genetic changes in the cancer cells has been provided. The DBCG 89-D trial showed that substitution of methotrexate in CMF with epirubicin lead to a reduction of 25% in overall survival [Mouridsen ASCO 1999]. Retrospectively it could be shown that this effect mainly is restricted to patients who harbor amplification or deletion of the TOP2A gene in their tumors [Knoop 2003]. The TOP2A gene encodes topoisomerase IIa that is the primary target of anthracyclines (e.g. epirubicin and doxorubicin).
TopoIIa is involved in DNA replication, transcription, and translation. TopoIIa introduces a temporary double DNA break, which allows the uncoiling of the DNA double helix. Following binding of epirubicin to topoIIa, the topoIIa complex is stabilized and the DNA break becomes irreversible [J M Berger 1996]. Anthracyclines are active during all phases of the cell cycle, but are most dynamic in proliferating cells [A J Coukell 1997].
In randomized trials, the survival benefit of adjuvant anthracycline-based chemotherapy seems to be restricted to ERBB2-positive patients [H B Muss 1994; A D Thor 1998; S Paik 1998; P C Clahsen 1998; S Paik 2000; A Di Leo 2001]. The gene encoding topoIIa (TOP2A) is situated close to ERBB2 and is often co-amplified with ERBB2. It has been suggested that the sensitivity to anthracycline-based chemotherapy in ERBB2-positive patients might be explained by the co-amplification of TOP2A and thereby increased expression of TopoIIa, the cellular target for anthracyclines [T A Järvinen 2000]. More recently, it has been shown that TOP2A gene copy number changes (CNC), both deletions and amplifications, are predictive to the outcome of anthracycline-based chemotherapy [Coon 2002; Di Leo 2002; Park 2003; Knoop, 2003; Harris 2004].
An important mode of action for many cytotoxic agents is the induction of DNA damage, and DNA repair can lead to general resistance. The main mode of action of Cyclophosphamide is generation of DNA cross-links that can be repaired directly or by nucleotide excision repair. Nucleotide excision repair is also central to the exchange of fluorouracil metabolites. XPD (Xeroderma Pigmentosum gene D) is involved both in the global DNA repair and the transcription dependent version of nucleotide excision repair [Reed 1998; Readon 1999] and ERCC1 (excision repair cross-complementing 1) is another critical element in this mechanism [Jiang 1999].
Thymidylate synthase (abbreviated TS for the protein and TYMS for the gene) use 5,10-Methylenetetrahydrofolat (MTHF) as a cofactor in the regulation of dTMP (thymidine monophosphat) that is critical to DNA replication and repair. The prognostic and predictive value of TS expression in tumor tissue has been the subject of numerous studies and high TS protein seems to predict resistance to TS inhibitors in patients with advanced colon cancer (Leichman 1998; Lenz 1998; Paradiso 2000). The results are less clear in the adjuvant setting where genetic markers seem to have predictive value (Watanabe 2001; Iacopetta 2001) while the IHC results are contradicting (Takenoue 2000; Edler 2002; Allegra 2003). Most likely dihydrofolate reductase (DHFR) is the main cellular target of methotrexate, and amplification of DHFR has often been correlated to resistance to both methotrexate and fluorouracil in experimental systems (Chu 1990; Banerjee 2002). The importance of thymidine phosphorylase (TP alias of ECGF1), dihydropyrimidine dehydrogenase (DPD alias of DPYD), Thymidine kinase (TK) and Methylenetetrahydrofolate reductase (MTHFR) has not yet been clarified.
DNA Synthesis Inhibitors
The synthesis and normal function of DNA and RNA is inhibited by the fluoropyrimidine 5-fluorouracil (5-FU) through inhibition of essential enzymes, primarily thymidylate synthase (TS), in the biosynthetic process and from incorporation of fluoronucleotides into the two macromolecules. Several inactive prodrugs of 5-FU have been developed, which are designed to allow intact absorption through the gastrointestinal canal and which are subsequently converted into 5-FU by metabolic transformation in the tumor or the liver. Several oral fluoropyrimidines are in development and the activity of capecitabine and UFT (uracil/Ftorafur) have been demonstrated in phase II, but results from phase III trials are awaited [M Malet-Martino 2002]. The tumor selectivity and conversion of capecitabine preferentially in tumor tissue have been demonstrated in a pharmacodynamic study, with a 3.2 fold higher FU concentration in tumor compared to normal tissue and 21 fold higher concentrations in tumor tissue compared to plasma [J Schüller 2000]. Cytotoxic chemotherapy with DNA synthesis inhibitors (as 5-FU) has demonstrated activity in a large range of malignancies and both in early and late stages of the disease. Activity has been observed both when 5-FU or analogs are given as single agents and in combinations with other agents. The importance of gene copy number changes as predictive markers has just recently been shown for Topoisomerase inhibitors. TOP2A gene copy number changes serve as an example of the importance of deletions and amplifications in predicting the outcome of anthracycline based treatment. Anthracyclines have been in clinical use since the sixties. Doxorubicin is produced by Streptomyces, and was in the beginning considered to be an antibiotic. Epirubicin is a semi-synthetic second-generation drug, with a more favorable therapeutic profile regarding cardio toxicity, stomatitis, and thrombopenia [D Ormrod 1999]. After 30 years of clinical use it became clear in the early nineties, that an inhibition of topoisomerase IIa protein (topoIIa) is the primary cytotoxic action of the anthracyclines [A V Gudkov 1993]. TopoIIa is involved in DNA replication, transcription, and translation. TopoIIa introduces a temporary double DNA break, which allows the uncoiling of the DNA double helix. Following binding of epirubicin to topoIIa, the topoIIa complex is stabilized and the DNA break becomes irreversible [J M Berger 1996]. Anthracyclines are active during all phases of the cell cycle, but are most dynamic in proliferating cells [A J Coukell 1997].
Drugs such as e.g. 5-Fluorouracil (5-FU) and Methotrexate interfere with key enzymatic steps in the nucleic acid syntheses. They inhibit essential biosynthetic processes and are incorporated into macromolecules such as DNA and RNA and thereby disrupt their normal function. 5-FU was introduced in 1957 and is still part of the most widely used drugs in adjuvant treatment of breast- and colon cancer. The first combination chemotherapy regimens for advanced breast cancer included antimetabolites and were introduced more than four decades ago [E M Greenspan 1963]. Following the early results of a randomized trial in advanced breast cancer [R G Cooper ASCO 1969; 10] methotrexate (MTX) and 5-FU were included in several later adjuvant trials [EBCTCG 1998].
The antimetabolites are early examples of rationally designed anticancer agents [C Heidelberger 1957]. The predominant view on the primary cellular action target of MTX is dihydrofolate reductase enzyme, and amplification of DHFR is one of the most common forms of methotrexate resistance observed in experimental systems [E Chu 1990]. Amplification of DHFR has also been associated with resistance to 5-FU in experimental systems [D Banerjee 2002]. Another potential explanation for the effect of MTX is inhibition of essential parts of the glucose transport mechanisms [K P Fung 1995].
The primary indications for use of 5-FU further to breast cancer are lung, prostate, colorectal, head & neck, pancreas, bladder, stomach, esophagus and liver.
The classification of tumors was until a decade ago often solitarily based on histology and more recently immunohistochemistry has been added. The staining pattern is however insufficient to reflect the underlying multiple molecular events characterizing the cancer cells viewed under the microscope. By surveying thousands of genes at once, using DNA arrays, it is possible to address the individual signature of every single tumor.
To improve the stratification of patients for the best treatment it would be of great benefit to the patient to select factors, which may predict the response of specific chemotherapeutic treatments. One therapeutic drug that is widely used in breast cancer as well as other types of cancer is 5-fluorouracil (5-FU).
It is thus highly desirable in the light of the aforementioned problems to develop means and methods for analyzing a patients ability to respond to a specific chemotherapeutic treatment, such as 5-FU-treatment, to increase the cancer patients wellbeing and to avoid unpleasant side effects in said cancer patients, such as e.g. breast cancer patients. Also, there is a need for identification of patients with tumors resistant to 5-FU treatment before the initiation of therapy, to avoid unpleasant side effects of said 5-FU treatment. In this respect, the present invention addresses those needs and interests.